Extracellular vesicle-mediated delivery to cells

Abstract

The invention concerns a loaded extracellular vesicle (EV) such as an exosome, wherein the EV has been loaded with a cargo molecule covalently or non-covalently coupled to a cell penetrating polypeptide (resulting in a “binding complex”), and the cargo molecule or binding complex has been internalized by, or is associated with, the EV. Another aspect of the invention concerns a method for loading an EV with a cargo molecule, comprising contacting the EV with the binding complex, wherein the binding complex becomes internalized by, or associated with, the EV. Another aspect of the invention concerns a method for delivering a cargo molecule into a cell in vitro or in vivo, comprising administering a loaded EV to the cell in vitro or in vivo, wherein the loaded EV is internalized into the cell, and wherein the loaded EV comprises the cargo molecule covalently or non-covalently bound to a cell penetrating polypeptide.

We claim: 
     
         1 . A method for loading an extracellular vesicle (EV) with a cargo molecule, comprising contacting the EV with a binding complex, wherein the binding complex comprises the cargo molecule and a cell penetrating polypeptide (CPP) covalently or non-covalently coupled to the cargo molecule, and wherein the binding complex becomes internalized by, or associated with, the EV. 
     
     
         2 . The method of  claim 1 , wherein the CPP is covalently coupled to the cargo molecule by a disulfide bond, an amide bond, a chemical bond formed between a sulfhydryl group and a maleimide group, a chemical bond formed between a primary amine group and an N-Hydroxysuccinimide (NETS) ester, a chemical bond formed via Click chemistry, or other covalent linkage. 
     
     
         3 . The method of  claim 2 , wherein the CPP is covalently coupled to the cargo molecule by a cleavable linker. 
     
     
         4 . The method of  claim 3 , further comprising uncoupling the cargo molecule and CPP of the binding complex by cleaving the cleavable linker after the binding complex becomes internalized by, or associated with, the EV. 
     
     
         5 . The method of  claim 1 , wherein the cargo molecule is selected from among a small molecule, macromolecule, protein, polypeptide, nucleic acid, antibody or antibody-fragment, lipid, metabolite, lipoprotein, carbohydrate, or glycoprotein. 
     
     
         6 . The method of  claim 1 , wherein the cargo molecule is a detectable agent or medical imaging agent, or is attached to a detectable or medical imaging agent, such as a fluorescent compound to serve as a marker, dye, tag, or reporter. 
     
     
         7 . The method of  claim 1 , wherein the EV further comprises a targeting agent that targets the EV to a cell type, organ, or tissue. 
     
     
         8 . The method of  claim 1 , wherein the CPP is one listed in Table 2 or Table 11. 
     
     
         9 . The method of  claim 1 , wherein the CPP is selected from among the following: Tat, Antennapedia, VP22, CaP, YopM, Artificial protein B1, 30Kc19, engineered+36 GFP, naturally supercharged human protein, and gamma-AApeptide. 
     
     
         10 . The loaded EV produced by the method of  claim 1 . 
     
     
         11 . A loaded extracellular vesicle (EV), comprising a cargo molecule and a cell penetrating polypeptide (CPP), wherein the cargo molecule has been internalized by, or associated with, the EV. 
     
     
         12 . The loaded EV of  claim 11 , wherein the loaded EV comprises a binding complex, wherein the binding complex comprises the cargo molecule and a CPP covalently or non-covalently coupled to the cargo molecule, and wherein the binding complex has been internalized by, or associated with, the EV. 
     
     
         13 . The loaded EV of  claim 12 , wherein two or more CPP are covalently or non-covalently coupled to the cargo molecule. 
     
     
         14 . The loaded EV of  claim 12 , wherein the CPP is coupled to the cargo molecule by a cleavable linker. 
     
     
         15 . The loaded EV of  claim 11 , wherein the cargo molecule is selected from among a small molecule, macromolecule, protein, polypeptide, nucleic acid, antibody or antibody-fragment, lipid, metabolite, lipoprotein, carbohydrate, or glycoprotein. 
     
     
         16 . The loaded EV of  claim 11 , wherein the CPP is one listed in Table 2 or Table 11. 
     
     
         17 . The loaded EV of  claim 11 , wherein the CPP is selected from among the following: Tat, Antennapedia, VP22, CaP, YopM, Artificial protein B1, 30Kc19, engineered +36 GFP, naturally supercharged human protein, and gamma-AApeptide. 
     
     
         18 . A method for delivering a cargo molecule into a cell in vitro or in vivo, comprising administering a loaded extracellular vesicle (EV) to the cell in vitro or in vivo, wherein the loaded EV comprises the cargo molecule and a cell penetrating polypeptide (CPP) wherein the cargo molecule has been internalized by, or associated with, the EV, and wherein the loaded EV is internalized into the cell. 
     
     
         19 . The method of  claim 18 , wherein the loaded EV comprises a binding complex, wherein the binding complex comprises the cargo molecule and the CPP covalently or non-covalently coupled to the cargo molecule, and wherein the binding complex has been internalized by, or associated with, the EV. 
     
     
         20 . The method of  claim 19 , wherein the loaded EV is administered to the cell in vivo by administering the loaded EV to a subject having the cell.